The study of neurological mutations offers a powerful approach to the identification of genes that regulate mammalian brain development. Work in our laboratory has established that the weaver gene regulates a critical step in neuronal differentiation, blocking the progression of immature granule neurons through the early steps of differentiation, including neurite production and migration. The proposed research will combine the tools of cell and molecular biology to further characterize the weaver gene. To understand earlier events in the development of weaver granule cells that map near the weaver locus, genetic markers will be used to identify weaver embryos by a simple PCR analysis, allowing developmental analysis of the midline aspect of the mid/hindbrain territory. In the proposed program, we will examine whether weaver cells show a deficit in the expression of genes encoding DNA binding proteins that mark the cerebellar primordium, and the generation of specific classes of cerebellar neurons by expression of cellular antigen markers within the midline region of the cerebellar anlage. To identify the ligand revealed by the weaver deficit, we will pursue a biochemical approach, fractionating membrane material from GC-B6 cells and using in vitro assays to identify the activity. Peptide sequence for candidate proteins will be obtained in order to obtain cDNAs encoding these ligands from granule cell cDNA libraries. Finally, the proposed research will use in vitro assays to clone the weaver gene. To refine the chromosomal location of weaver, new inter-species backcrosses will be carried out, after which a contiguous set of yeast artificial chromosomes (YACs) containing DNA from the region containing the weaver locus will be constructed. Transfer of DNAs from these YACs to immortalized granule cells will provide a reagent for the rapid location of the weaver gene on the YAC contig, by assaying the ability of transfected cells to rescue weaver granule cell differentiation in complementation assays. Candidate genes will be isolated and screened for the weaver mutation. The proof that the candidate cDNA encodes the weaver gene will be tested in the in vitro complementation assays and ultimately in transgenic mice.